Interstitial adenosine and cellular metabolism during beta-adrenergic stimulation of the in situ rabbit heart.

OBJECTIVE: Adenosine, derived from hydrolysis of 5'-AMP, may be involved in coupling coronary blood flow to cardiac function and metabolism. The purpose of this study was to measure interstitial fluid (ISF) adenosine and 5'-AMP levels, and cytosolic 5'-AMP in in situ rabbit heart during beta-adrenergic stimulation. METHODS: Isoproterenol was infused into open chest rabbits (n = 7) at 1 and 4 micrograms.kg-1.min-1. Left ventricular ISF adenosine and 5'-AMP levels were measured using microdialysis, and energy metabolism simultaneously monitored using 31P-NMR spectroscopy. RESULTS: Graded beta-stimulation increased heart rate (by 50% and 70%), arterial pulse-pressure (by 55% and 45%), and the rate-pressure product (by 45% and 70%). Dialysate [adenosine] increased 300-400% from a control value of 0.44 +/- 0.13 microM. Dialysate [5'-AMP] increased 200% from a control value of 0.94 +/- 0.22 microM. Cytosolic [ATP], pH and free [Mg2+] remained stable, whereas [PCr] declined by 10-20%. Free cytosolic [5'-AMP] increased 300-400% from a control value of 0.40 microM. Competitive inhibition of ecto-5'-nucleotidase with alpha,beta-methylene-ADP (0.6 mg.kg-1.min-1) significantly reduced ISF adenosine (and enhanced ISF 5'-AMP) during beta-stimulation, but not under basal conditions. CONCLUSIONS: Beta-adrenergic stimulation increases ISF adenosine levels and depresses bioenergetic state in in situ rabbit heart without altering [ATP], pH or [Mg2+], indicating an absence of "demand" ischemia. ISF adenosine originates primarily from cytosolic 5'-AMP under basal conditions whereas increased adenosine during beta-stimulation appears to occur via hydrolysis of both ISF and cytosolic 5'-AMP. ISF adenosine levels achieved during stimulation are appropriate for stimulation of cardiovascular A1 and A2 receptors.

Effects of xanthine amine congener on hypoxic coronary resistance and venous and epicardial adenosine concentrations.

OBJECTIVE: The aim was to define the contributions of interstitial and vascular adenosine in regulating coronary vascular resistance during hypoxia. To help in the assessment of adenosine in the vasodilator response, a potent adenosine receptor antagonist, xanthine amine congener (XAC), was used to block adenosine receptors. METHODS: Seven isolated guinea pig hearts were perfused at constant flow with Krebs buffer. Coronary vascular resistance was determined during normoxia (95% O2) and mild hypoxia (60% O2) in the absence or presence of 200 or 400 nM XAC. Interstitial fluid was sampled by the epicardial disc technique and the interstitial concentration of XAC (ISF[XAC]) was determined directly by a radioreceptor assay or as tritiated XAC. Venous and epicardial concentrations of adenosine were determined by high performance liquid chromatography. In six additional experiments, the vasodilator effect of 1 microM intracoronary adenosine was measured in the absence or presence of 100 or 200 nM XAC. RESULTS: Mild hypoxia decreased coronary resistance by 37 (SEM 4)% in the absence of XAC and 26(5)% or 17(4)% in the presence of 200 or 400 nM XAC, respectively. ISF[XAC] rapidly equilibrated with [XAC] in the arterial perfusate or venous effluent. XAC 400 nM markedly increased (p < 0.05) the hypoxic levels of venous and epicardial fluid adenosine from 49(19) and 251(42) nM to 75(11) and 495(48) nM, respectively. XAC 100-200 nM almost completely prevented the vasodilatation induced by 1 microM intracoronary adenosine. CONCLUSIONS: Adenosine mediates at least 54% of hypoxic vasodilatation. XAC rapidly equilibrates within the myocardial interstitial space and, as a result of blocking adenosine receptors, increases interstitial and venous adenosine concentrations. Increases in interstitial adenosine may partially overcome the adenosine receptor blockade by XAC, thereby reducing the effectiveness of XAC in attenuating the hypoxic vasodilatation. XAC attenuates intracoronary adenosine induced vasodilatation (mediated by endothelial adenosine receptors) much more effectively than it attenuates hypoxic vasodilatation, underscoring the minimal role played by the endothelial receptors in hypoxic vasodilatation.

Adenosine and insulin mediate glucose uptake in normoxic rat hearts by different mechanisms.

The effect of adenosine (ADO) and its interaction with insulin (I) on myocardial glucose uptake was evaluated in the normoxic isolated rat heart using 2-[3H]deoxyglucose. Isovolumic hearts were perfused at constant flow with a nonrecirculating bicarbonate buffer containing 5.5 mM glucose as the sole substrate. After a 30-min equilibration period, the glucose and extracellular ([14C]sucrose) tracers were infused for 15 min before initiation of the 15-min experimental period. Both 100 microM ADO and 4 mU/ml I significantly increased glucose uptake (GU) compared with control values (in mumol.min-1 x g-1: ADO = 0.34 +/- 0.03, I = 0.44 +/- 0.03, control = 0.23 +/- 0.02; P < 0.05). In combination, ADO and I produced an additive increase in GU (0.54 +/- 0.03; P < 0.05 vs. control). The mechanism of enhanced GU by ADO and I was investigated with the glucose uptake inhibitors phloridzin (PZ) and phloretin (PT), each of which has a unique site of action on the cell membrane. ADO-mediated GU was completely blocked by 3 mM PZ (ADO + PZ = 0.20 +/- 0.01; P = NS vs. control), but I-stimulated GU was unaffected (I + PZ = 0.38 +/- 0.03; P = NS vs. I). Only GU attributable to ADO was blocked by PZ infused with ADO and I (ADO + I + PZ = 0.43 +/- 0.03; P = NS vs. I). Both ADO- and I-mediated GU were inhibited by 100 microM PT.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic and functional responses of immature and mature rabbit hearts to hypoperfusion, ischemia, and reperfusion.

Relationships between contractile function and cytosolic metabolism were examined in mature and immature rabbit hearts during graded hypoperfusion, global ischemia, and reperfusion. Differences in basal contractile function and metabolic demand in immature and mature hearts were paralleled by differences in energy substrate indexed by the phosphorylation potential and phosphocreatine (PCr) levels but not by differences in ATP, pH, or inorganic phosphate (Pi). During graded hypoperfusion, contractility declined comparably in both age groups. Relative changes in [ATP], [PCr], and [Pi] were similar in both groups, whereas pH declined to a greater extent in mature hearts, and [Mg2+] declined to a greater extent in immature hearts. Contractile function correlated most consistently with [Pi] and supports the notion that Pi (and H+) are primary determinants of function in underperfused immature and mature hearts. Aerobic efficiency declined in immature but not mature hearts during hypoperfusion, reflecting an improved ratio of ATP formation relative to ATP hydrolysis in the hypoperfused immature myocardium. Finally, the enhanced contractile recovery from global ischemia in immature compared with mature hearts (to 93 +/- 4 vs. 79 +/- 2% of basal function) was unrelated to recovery of ATP, Pi, pH, Mg2+, [ATP]/[ADP].[Pi], or free-energy change of ATP hydrolysis. Age-related differences in maintenance of ATP may be related to enhanced metabolic activation of glycolysis coupled with better buffering of pH and an improved match between ATP hydrolysis and ATP formation in immature heart.

Changes in extracellular adenosine during chemical or electrical brain stimulation.

The purpose of this study was to determine the changes in adenosine and adenosine metabolites during graded electrical stimulation or kainic acid-induced activation and to assess the role of adenosine in the cerebral blood flow (CBF) response to increased brain activity. A modified brain microdialysis technique was used to sample cerebral interstitial fluid (ISF), deliver drugs locally to the brain, electrically stimulate the brain, and measure local CBF (H2 clearance). Microdialysis probes were implanted bilaterally in the caudate nuclei of ketamine-anesthetized rats. Graded electrical stimulation at 5, 15, and 30 Hz increased dialysate adenosine 1.5-fold, 2.3-fold, and 4.7-fold, respectively. Local infusion of kainic acid, an agonist of the excitatory amino acid neurotransmitter glutamate, produced a transient increase (2-fold) in dialysate adenosine and sustained increases in dialysate inosine (2-fold), hypoxanthine (4-fold) and CBF (2.4-fold). When the adenosine receptor antagonist 8(p-sulphophenyl)-theophylline (SPT, 10(-3) M) was co-administered with kainic acid, CBF increased only 1.6-fold, while the increase in dialysate adenosine was augmented by 40%. These data demonstrate that ISF adenosine increases during brain activation and suggest that adenosine contributes to active hyperemia in the brain.

Myocardial adenosine, flow, and metabolism during adenosine antagonism and adrenergic stimulation.

Relationships between interstitial transudate adenosine and coronary flow and between global adenosine formation and cytosolic metabolism were examined in constant-pressure perfused guinea pig hearts during norepinephrine (NE) stimulation and adenosine antagonism with 10 microM 8-phenyltheophylline. Basal coronary flow was 5.7 ml.min-1 x g-1, and transudate and venous adenosine levels were approximately 0.26 and 0.06 microM, respectively. During 10 min of NE stimulation (15 nM), coronary flow and adenosine levels increased, the phosphocreatine-to-inorganic phosphate ratio ([PCr]/[Pi]) declined, and ATP and pH remained stable. Despite phasic release of adenosine, coronary flow correlated dose dependently with transudate adenosine, and adenosine release was inversely related to [PCr]/[Pi] under all conditions. 8-Phenyltheophylline infusion attenuated functional hyperemia by approximately 40%, enhanced the fall in [PCr]/[Pi], and potentiated elevations in transudate and venous adenosine. Similar results and correlations were obtained in hearts perfused at a constant-flow of 5.7 ml.min-1 x g-1, although stimulated adenosine levels and metabolic changes were greater and contractile responses smaller. These data indicate that: 1) endogenous adenosine plays a primary role in functional hyperemia in perfused guinea pig heart; 2) global adenosine formation appears related to phosphorylation status; and 3) adenosine receptor antagonism enhances metabolic disturbances during adrenergic stimulation and markedly potentiates adenosine release, indicating that the functional effects of antagonists may significantly underestimate the dilatory role of endogenous adenosine.

Changes in work rate to oxygen consumption ratio during hypoxia and ischemia in immature and mature rabbit hearts.

This study was designed to evaluate the relative response of myocardial efficiency to reduced oxygen supply (hypoxia and ischemia) in immature and mature isolated rabbit hearts. Hearts were subjected to either 15 min of hypoxia (60% or 30% O2) or reductions in coronary flow to 75%, 50%, 25%, and 15% of basal flow followed by 12 min of total global ischemia and 15 min of reperfusion. In order to examine changes in cardiac efficiency, we utilized the ratio of isovolumic contractile function (rate-pressure product) to myocardial oxygen consumption (RPP/MVO2). Under basal conditions, immature hearts displayed lower aortic pressure. RPP, coronary resistance and RPP/MVO2. Moderate hypoxia (60% O2) resulted in similar reductions in RPP and MVO2 in both age groups, with RPP/MVO2 remaining unchanged. During severe hypoxia, RPP/MVO2 increased significantly in mature hearts but not in immature hearts (P < 0.05). Underperfusion produced greater reductions in RPP and heart rate, whereas reperfusion after ischemia resulted in greater recovery of RPP, dP/dt and MVO2 in immature compared to mature hearts. When oxygen supply was limited by reductions in coronary perfusion. RPP/MVO2 tended to increase in mature hearts, whereas the ratio declined significantly in immature hearts. These data demonstrate that, in this model, a reduction in oxygen supply by hypoxia or hypoperfusion decreases efficiency in immature hearts, but increases efficiency in mature hearts under the same conditions.

Relative responses to luminal and adventitial adenosine in perfused arteries.

Responses to luminal and adventitial adenosine were compared in perfused rabbit central ear arteries. Perfused arteries precontracted with 0.5 microM norepinephrine relaxed dose dependently but asymmetrically to luminal and adventitial adenosine. Arteries were more responsive to luminal adenosine in the 0.1- to 1.0-microM range, but they were more responsive to adventitial adenosine at doses > 10 microM. Alternatively, 2-chloroadenosine, a metabolically stable and poorly transported analogue, was equipotent when applied luminally or adventitially. Endothelial damage reduced sensitivity and response asymmetry to luminal and adventitial adenosine. This was consistent with reduced responses to adenosine in luminally rubbed arterial ring segments. Transport inhibition (10 microM dipyridamole) enhanced arterial reactivity to luminal and adventitial adenosine and reduced response asymmetry but was without effect on responses to 2-chloroadenosine. A comparison of the inhibitory effectiveness of adventitial and luminal 8-phenyltheophylline revealed that adventitial antagonist was approximately threefold more effective in inhibiting responses to adventitial adenosine than luminal antagonist (P < 0.05). This "side-dependent" difference was reduced by prolonged antagonist incubation or endothelial removal. The data indicate that adenosine relaxes ear arteries by activation of smooth muscle [half-maximum effective concentration (ED50) approximately 11 microM] and endothelial (ED50 approximately 2 microM) receptors. Nevertheless, a sensitive endothelial-dependent response does not consistently enhance responses to luminal adenosine in perfused arteries. This appears to be attributable to relative differences in the smooth muscle and endothelium-dependent components of the dilator response and transvascular concentration gradients for luminally and adventitially applied adenosine. A transendothelial diffusion barrier also reduces the ability of luminally applied antagonists to inhibit responses to adventitial adenosine.

Inhibition of adenosine metabolism increases myocardial interstitial adenosine concentrations and coronary flow.

We employed an isolated guinea-pig heart model perfused at constant pressure (70 cmH2O) to test the hypothesis that inhibition of adenosine metabolism increases interstitial adenosine concentrations (as measured with epicardial discs) and coronary flow. Iodotubercidin (ITU, 1 microM) and EHNA (erythro-9-[2-hydroxy-3-nonyl] adenine, 5 microM) were used to inhibit adenosine kinase and deaminase, respectively during control conditions and during metabolic stimulation with 1 microM isoproterenol. The adenosine receptor blocker 8-phenyltheophylline (8-PT) was used during control conditions to assess whether the response seen was adenosine specific. ITU plus EHNA decreased heart rate (202 +/- 10 to 136 +/- 11 beats/min) and increased coronary flow (8.2 +/- 0.3 to 12.4 +/- 0.9 ml/min/g) without a change in MVO2, developed pressure or dP/dt. ITU plus EHNA increased adenosine concentrations in epicardial fluid (0.24 +/- 0.07 microM to 1.02 +/- 0.09 microM) and venous effluent (40 +/- 3 nM to 262 +/- 32 nM) during control conditions, and adenosine release increased from 389 +/- 96 pmols/min/g to 3480 +/- 365 pmols/min/g. 8-PT infusion reversed the effects on heart rate and coronary flow and resulted in a persistent elevation of epicardial fluid adenosine concentrations. During metabolic stimulation with 1 microM isoproterenol, ITU plus EHNA significantly limited the increase in heart rate and ventricular developed pressure and dP/dt while coronary flow increased to a significantly greater extent. Myocardial oxygen consumption was similar during metabolic stimulation between the two groups (vehicle vs. ITU plus EHNA). Epicardial fluid adenosine concentration in the vehicle-treated group increased from 0.17 +/- 0.3 microM to 0.34 +/- 0.02 microM at 15 min of isoproterenol stimulation whereas it increased from 1.10 +/- 0.02 microM to 2.90 +/- 0.46 microM in the ITU plus EHNA-treated group. Inhibition of adenosine metabolism during metabolic stimulation significantly increased venous adenosine concentrations and adenosine release and reduced inosine and hypoxanthine release proportionately. The release of adenosine+inosine+hypoxanthine was unchanged. Inhibition of adenosine metabolism provides evidence supporting the hypothesis that adenosine plays a role in regulating coronary vascular resistance as well as influencing heart rate and ventricular inotropy.

Effect of adenosine deaminase on cardiac interstitial adenosine.

Adenosine deaminase was infused into isolated perfused guinea pig hearts to determine its effect on myocardial adenosine levels. The enzyme was administered during constant coronary flow perfusion at 6.11 +/- 0.36 ml.min-1.g-1. Venous adenosine was measured in samples of pulmonary artery effluent; epicardial and endocardial adenosine were measured with the porous nylon disk technique. Infusion of adenosine deaminase at 2.4 and 4.8 U/ml produced adenosine deaminase activity of 0.92 +/- 0.09 and 2.33 +/- 0.15 U/ml, respectively, in epicardial fluid and 1.93 +/- 0.28 and 4.84 +/- 0.47 U/ml, respectively, in endocardial fluid. Aortic pressure was unchanged by infusion of adenosine deaminase at both infusion rates. Adenosine deaminase (data from both infusion rates pooled) reduced epicardial adenosine from 0.327 +/- 0.028 to 0.139 +/- 0.022 microM, endocardial adenosine from 4.61 +/- 0.42 to 1.64 +/- 0.20 microM, and venous adenosine from 0.017 +/- 0.02 to 0.003 +/- 0.001 microM. The data indicate that infused adenosine deaminase reaches the epicardial and endocardial interstitial fluid (ISF) compartments. The absence of any effect on coronary pressure suggests that adenosine may not be involved in resting basal coronary tone. The presence of significant residual adenosine despite adenosine deaminase infusion indicates that adenosine production in the unstressed isolated guinea pig heart exceeds the degradative capacity of infused adenosine deaminase. Previous studies in which it was assumed that almost all of the endogenous adenosine is inactivated by the infusion of adenosine deaminase should be reevaluated in light of these observations.

Sciatic nerve stimulation does not increase endogenous adenosine production in sensory-motor cortex.

Adenosine participates in the coupling of cerebral blood flow to oxygen consumption in the brain during such stimuli as hypoxia, ischemia, and seizures. It has been suggested that it also participates in the regulation of cerebral blood flow during somatosensory stimulation, a condition during which cerebral blood flow and oxygen consumption appear to be uncoupled. Interstitial adenosine was estimated by the microdialysis technique and cerebral blood flow was measured by hydrogen clearance in the hindlimb sensory-motor cortex during sciatic nerve stimulation. Cerebral blood flow increased from 102 to 188 ml min-1 100 g-1 (p less than 0.001) in the cortex contralateral to the stimulated leg without an associated increase in interstitial adenosine (baseline 0.624 microM, stimulation 0.583 microM). Infusion of the adenosine antagonist 8-sulfophenyltheophylline failed to block an increase in cerebral blood flow during central sciatic nerve stimulation, but decreased basal cerebral blood flow (69 ml min-1 100 g-1). These results suggest that adenosine does not mediate changes in cerebral blood flow during somatosensory stimulation, but may participate in the regulation of cerebral blood flow in the basal state.

Competitive inhibition of nitric oxide synthase prevents the cortical hyperemia associated with peripheral nerve stimulation.

With combined microdialysis and hydrogen clearance techniques for simultaneous local delivery of drugs and blood-flow measurement in the rat hindlimb sensory-motor cortex, we examined the role of nitric oxide in cerebral blood-flow regulation during sciatic nerve stimulation. Infusion of 1 mM nitric oxide synthase antagonist, N eta-nitro-L-arginine methyl ester (L-NAME), blocked the cortical blood-flow response to sciatic nerve stimulation (152 +/- 43 ml.min-1.100 g-1 of tissue in controls and 73 +/- 11 ml.min-1.100 g-1 in the presence of L-NAME; P less than 0.05). Addition of 10 mM L-arginine to the dialysate containing L-NAME partially restored the hyperemic response to nerve stimulation (125 ml.min-1.100 g-1). L-NAME also produced a decrease in baseline cerebral blood flow when compared with the control (66 +/- 14 ml.min-1.100 g-1 vs. 93 +/- 25 ml.min-1.100 g-1). We conclude that nitric oxide from activated neurons participates in the local regulation of cortical blood flow in response to sciatic nerve stimulation and also in the maintenance of basal cortical blood flow.

Heterogeneity and sampling volume dependence of epicardial adenosine concentrations.

Rapid steady-state estimates of interstitial fluid (ISF) adenosine concentrations (ADOi) in the left ventricular epicardium of anesthetized dogs were obtained by the epicardial porous disc (EPD) method described herein. Because of the high temporal and spatial resolution of this method, it was ideally suited to test the hypothesis that ADOi may vary in these domains. Variance in steady-state EPD solute concentrations was quantified statistically by the coefficient of variation (CV = standard deviation/mean), which we used as an index of heterogeneity. A significant temporal variation in steady-state EPD adenosine concentrations was observed when samples were sequentially collected from one epicardial location (CV = 42.9 +/- 3.5%). When steady-state sample pairs (n = 45) were collected simultaneously from two distinct epicardial locations, a 2.6 +/- 0.3-fold mean difference in their respective adenosine concentrations was measured. About 25% of this variation was inherent in procedural methodology, based on the variability of steady-state EPD concentrations of extracellularly-equilibrated 14C sucrose (CV = 12.7 +/- 1.2%) and the variability of steady-state concentrations of both solutes measured using in vitro preparations (mean CV = 9.7 +/- 1.2%). Thus, we contend that endogenous myocardial ISF adenosine is temporally and perhaps spatially heterogeneous. Our estimates of steady-state ADOi obtained with the EPD method ranged from 0.47 to 0.99 microM. Using modifications of the EPD technique and the epicardial chamber, we also demonstrated that the adenosine concentration in 'steady-state' epicardial samples is reduced when the volume/surface area ratio of the sample buffer is increased. We hypothesize that sampling-induced decreases in steady-state ADOi underlie these observations, because losses of ISF adenosine to high volumes of sample buffer can be greater than the myocardial cells are capable of replacing. However, with the very low volume/surface area ratio of a single EPD (7.5 microliters/cm2), steady-state ADOi may remain constant during sampling, allowing for accurate determinations of ADOi with this method.

Protective effects of adenosine in myocardial ischemia.

Adenosine is released from the myocardium in response to a decrease in the oxygen supply/demand ratio, as is seen in myocardial ischemia; its protective role is manifested by coronary and collateral vessel vasodilation that increase oxygen supply and by multiple effects that act in concert to decrease myocardial oxygen demand (i.e., negative inotropism, chronotropism, and dromotropism). During periods of oxygen deprivation, adenosine enhances energy production via increased glycolytic flux and can act as a substrate for purine salvage to restore cellular energy charge during reperfusion. Adenosine limits the degree of vascular injury during ischemia and reperfusion by inhibition of oxygen radical release from activated neutrophils, thereby preventing endothelial cell damage, and by inhibition of platelet aggregation. These effects help to preserve endothelial cell function and microvascular perfusion. Long-term exposure to adenosine may also induce coronary angiogenesis.

Myocardial adenosine formation during hypoxia: effects of ecto-5'-nucleotidase inhibition.

Release of adenosine and AMP into epicardial fluid and coronary venous effluent of isovolumic guinea-pig hearts was examined during normoxic (95% O2) and hypoxic (30% O2) perfusion with and without the ecto-5'-nucleotidase inhibitor alpha,beta-methylene adenosine diphosphate (AOPCP)*. Normoxic epicardial and venous adenosine levels were 221 +/- 27 and 67 +/- 11 nM, respectively, in untreated hearts. During 15 min of hypoxia, epicardial and venous adenosine levels increased in a phasic manner, reaching maximal values of 498 +/- 32 and 441 +/- 43 nM, respectively, during the initial 5 min of hypoxia. Epicardial and venous adenosine levels then declined slightly during the subsequent 10 min to 332 +/- 33 and 224 +/- 34 nM, respectively. Infusion of 50 microM AOPCP significantly reduced venous adenosine levels during normoxia (less than 50% of control), but was without effect on normoxic epicardial adenosine. Epicardial and venous adenosine levels increased during hypoxia with AOPCP but the increases were lower than those for untreated hypoxic hearts. Epicardial and venous adenosine levels recovered to baseline levels following 30 min of reoxygenation in both groups. Epicardial and venous AMP levels were elevated by AOPCP treatment during normoxia and hypoxia. Coronary vascular resistance decreased during hypoxia but the decline in resistance was less in AOPCP treated hearts. It is concluded that whereas basal interstitial adenosine levels appear to be independent of ecto-5'-nucleotidase activity, the hypoxic increase in interstitial adenosine is partially derived from an AOPCP sensitive ecto-5'-nucleotidase. Venous adenosine appears to be significantly dependent on ecto-5'-nucleotidase activity during normoxia and hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelial-dependent and -independent responses in the thoracic aorta during endotoxic shock.

Endotoxic shock is characterized by a variety of hemodynamic disturbances which result in tissue hypoperfusion. There is some evidence for endothelial damage caused by endotoxin. The present study addressed the hypothesis that vascular responsiveness to endothelial-dependent vasodilators is altered in endotoxic shock. Dose-response relationships for an endothelial-dependent vasodilator, acetylcholine, and an endothelial-independent vasodilator, adenosine, were determined in guinea pig aortic rings. Rings were examined from either control (untreated) animals or from animals given Escherichia coli endotoxin (4 mg/kg, i.p.) 16 hr prior to functional studies. Dose-response relationships to adenosine were similar in aortic rings from control and shocked animals. However, response to acetylcholine were attenuated by 30% (P less than .05) in the shocked group. To distinguish between a direct, acute effect of endotoxin versus effects produced by systemic changes that occur during shock, rings were isolated from untreated animals and incubated with endotoxin in vitro for 30 min prior to and during dose-response measurements. Incubation with endotoxin caused no change in aortic responses to adenosine or acetylcholine. Electron microscopy revealed a separation of the endothelium from the internal elastic lamina and an increase in inter-endothelial gaps in rings isolated from shocked animals. These structural changes were not observed in rings from untreated animals or in rings incubated with endotoxin in vitro. We conclude that endothelial-dependent vasodilation is attenuated during endotoxic shock. The functional changes are correlated with ultrastructural alterations of the endothelium.

Effects of graded perfusion and isovolumic work on epicardial and venous adenosine and cytosolic metabolism.

Epicardial adenosine levels and venous adenosine release were measured in isovolumically contracting (ISO) and empty non-isovolumic (non-ISO) guinea-pig hearts subjected to graded perfusion (approximately 7.5, 5.5, 4.0, 2.0, and 1.0 ml/min/g). Myocardial metabolism was monitored using 31P-NMR spectroscopy. At flows of 5.5 ml/min/g or higher epicardial adenosine levels were stable and comparable in ISO and non-ISO hearts (approximately 160 nM). At flows of 4.0 ml/min/g or higher venous adenosine release was stable and comparable in ISO and non-ISO hearts (approximately 30 pmol/min/g). At lower flows, epicardial adenosine and venous adenosine release both increased and were significantly higher in ISO hearts, compared to non-ISO hearts, at each flow rate. Whereas epicardial adenosine increased linearly in ISO and non-ISO hearts at low flows, venous adenosine release stabilized in ISO hearts perfused at 1.0 ml/min/g. Epicardial adenosine, venous adenosine release, and log [ATP]/[ADP] [Pi] all displayed significant correlations with the O2 supply:demand ratio which were comparable in ISO and non-ISO hearts. Elevated levels of epicardial adenosine were linearly related to log [ATP]/[ADP] [Pi] and cytosolic [AMP] and these relationships were comparable in ISO and non-ISO hearts. Alternatively, changes in venous adenosine release did not display simple relationships with log [ATP]/[ADP] [Pi] and cytosolic [AMP] and they were not comparable in ISO and non-ISO hearts. The data indicate that: (i) myocardial adenosine formation increases only below a metabolic threshold corresponding to log [ATP]/[ADP] [Pi] = 5.0 and O2 supply:demand = 1.5 in ISO and non-ISO guinea-pig hearts; (ii) stimulated epicardial adenosine levels appear to be consistently related to changes in cytosolic metabolism below this threshold in ISO and non-ISO hearts; (iii) more complex relationships exist between venous adenosine release and myocardial metabolism during graded perfusion, possibly reflecting the variety of factors modulating venous adenosine release.

Metabolic correlates of adenosine formation in stimulated guinea pig heart.

Adenosine release into epicardial fluid and coronary effluent of isolated isovolumic guinea pig hearts was examined at baseline and after stimulation with norepinephrine (30 nM) during 31P-nuclear magnetic resonance spectroscopy to monitor myocardial metabolism. At baseline flow (9.6 +/- 0.3 ml.min-1.g-1), epicardial and venous adenosine concentrations were 154 +/- 40 and 17 +/- 5 nM, respectively. The phosphorylation potential (log[ATP]/[ADP][Pi]) and the phosphocreatine-inorganic phosphate ratio ([PCr]/[Pi]) were 5.26 +/- 0.04 and 8.5 +/- 0.7, respectively. Norepinephrine increased left ventricular pressure, heart rate, and myocardial O2 consumption rate by approximately 21, 70, and 45%, respectively, and increased epicardial and venous adenosine to 496 +/- 74 and 461 +/- 94 nM, respectively. Log-[ATP]/[ADP][Pi] and [PCr]/[Pi] declined to 4.57 +/- 0.06 and 1.9 +/- 0.3, respectively. Epicardial [AMP] increased from 54 +/- 13 to 123 +/- 24 nM. AMP was not detectable in the venous effluent. Coronary resistance correlated with epicardial and venous [adenosine] (r = 0.86 and 0.90). Epicardial and venous [adenosine] correlated with log[ATP]/[ADP][Pi], [PCr]/[Pi], and cytosolic [AMP]. Hence, interstitial adenosine is linked to cytosolic metabolism and may regulate coronary vascular resistance. Venous adenosine underestimates epicardial adenosine at baseline but more closely approximates epicardial adenosine during norepinephrine infusion.